Each vineyard has its own wine of microbiota, which affects the quality of the resulting wine. Separation and cultivation methods can be used to characterization and multiplying individual microorganisms. Based on the fermentation and H₂S production tests, the resulting consortium can be assembled, which can be used for fermentation, thereby promoting both the fermentation process and the terroir of the wine.

MEA+T Malt Extract Agar with Tetracycline (a broad-spectrum antibiotic against both gram-positive G+ and gram-negative G- bacteria). Only eukaryotic microorganisms grow on this medium.

Composition: agar – 15 g.L^-1, malt extract – 30 g.L^-1, mycological peptone – 5 g.L^-1, (manufacturer: Sigma Aldrich)

WLN: Wallerstein Nutrient Agar for counting and culturing yeast and bacteria.

Composition: agar – 20 g.L^-1, bromocresol green – 0.022 g.L^-1, calcium chloride – 0.125 g.L^-1, casein enzymic hydrolysate – 5 g.L^-1, dextrose – 50 g.L^-1 (manufacturer: Sigma Aldrich).

MRS (Agar according to DeMan, Rogosa, and Sharpe): Nutrient agar for the determination of lactic bacteria.

Composition: Agar – 12 g.L^-1, diammonium bicarbonate – 2 g.L^-1, potassium hydrogen phosphate – 2 g.L^-1, D (+) – glucose – 20 g.L^-1, magnesium sulphate – 0.1 g.L^-1, manganese sulphate – 0.05 g.L^-1, meat extract – 5 g.L^-1, sodium acetate – 5 g.L^-1, universal peptone – 10 g.L^-1, yeast extract – 5 g.L^-1. (manufacturer: Sigma Aldrich)

YPD (Yeast extract Peptone Dextrose) agar: Solid medium for yeast multiplication.

Composition: Bacteriological peptone – 20 g.L^-1, yeast extract – 10 g.L^-1, glucose – 20 g.L^-1, agar – 15 g.L^-1. (manufacturer: Sigma Aldrich)

ME (M-enterococcus) agar: Agar consists of tryptose; yeast extract; glucose; disodium hydrogen phosphate; sodium azide; 2, 3, 5-triphenyltetrazolium chloride; agar; and distilled or deionized water. (manufacturer: Sigma Aldrich)

BIGGY (Bismuth Sulphite Glucose Glycine Yeast Agar): Selective and differential medium with addition of bismuth salt for H₂S detection. Bismuth reacts with the resulting sulfane to form a precipitate that colours the agar below the colony.

Composition: Glucose – 10.0 g.L^-1, glycine – 10.0 g.L^-1, bismuth ammonium citrate – 5.0 g.L^-1, sodium sulphite – 3.0 g.L^-1, yeast extract – 1.0 g.L^-1, agar – 13.0 g.L^-1. (manufacturer: Sigma Aldrich)

The must of the Hibernal variety: This variety was developed in Germany in 1944 as a hybrid of Seibel 7053 (Chancellor) and Riesling. The must be clarified by sedimentation (after 24 hours). The turbidity value of the must after clarifying was approx. 400 NTU and this was not adjusted. The sugar content was 16 °NM, pH was 3.51, titratable acid content was 6.48 g.L^-1, and assimilable nitrogen content was 321 mg.L^-1.

The must of the Hibernal variety was fermented spontaneously. During this spontaneous fermentation, 20 mL of the matrix (must, fermentation must, wine) was taken at selected monitoring points (must, micro-sparkling, 4 and 8 vol. % ethanol, end of fermentation). The sample obtained was subsequently diluted using the so-called decimal series. From each dilution, 250 mL was pipetted onto Petri dishes with MEA+T, WLN, and MRS culture medium, and a microbiological rod smear was performed. The Petri dishes were then placed in a thermostat (30 °C; WLN and MEA+T – 3 days; MRS – 7 days).

At the end of the cultivation, the total number of microorganisms and individual colonies was enumerated. Based on a combined analysis of phenotypic characters (macroscopic and microscopic properties), a sampling point was selected (the most suitable was the micro-sparkling point - the diversity of technologically important microorganisms was evaluated – saccharomyces and non-saccharomyces yeasts and lactic bacteria) to serve as a source for consortium acquisition.

The aim was to determine the growth characteristics of individual isolates. Individual isolates of microorganisms were pre-cultured in standard media (yeast – YPD; lactic acid bacteria - MRS) with the following culture conditions: 30 °C; shaking 120 rpm; yeast 24 h; and lactic acid bacteria 72 h. The obtained cell suspensions were centrifuged (10 mins; 10 °C; 10,000 rpm), washed with saline solution, and then resuspended in the selected media (yeast – YPD, ME, YPD_mod; lactic acid bacteria – MRS, YPD, and YPD_mod) so that the resulting optical density value of the suspension was 0.2 at a wavelength of 600 nm.

The obtained suspension was then pipetted onto Bioscreen C culture plates (Oy Growth Curves Ab Ltd). Each arrangement (microorganism x medium) consisted of five repetitions to ensure the achievement of relevant results.

The culture conditions of the Bioscreen C device were set as follows: 30 °C; shaking every 3 mins; duration of one shaking cycle = 1 min; and the so-called wide band of wavelengths – WB (420 – 620 nm). The maximum growth rate μ (h^-1) was then calculated from the measured data, and the maximum optical density OD_MAX was determined. These data, along with the course of growth curves, served to assign culture media to individual isolates.

The MALDI TOF (matrix-assisted laser desorption/ionization coupled with time of flight mass spectrometry) method was used to identify microorganisms. It is a very accurate, simple method, able to determine high molecular weight substances, proteins, peptides, lipids, nucleic acids, carbohydrates (Huong et al., 2014).

An essential part of the MALDI TOF measurement was the preparation of fresh α-cyano-4-hydroxycinnamic acid. The organic solvent was prepared by mixing 500 μL of acetonitrile (100%), 475 μL of distilled water, and 25 μL of trifluoroacetic acid. Before use, 250 μL of organic solvent was added to the plastic tube. The contents of the tube were vortexed until the complete dissolution of the crystals. α-cyano-4-hydroxycinnamic acid was stored in the dark place and its preparation is ideal the day before the measurement.

The cultures were applied to the clean metal plate for MALDI TOF and the culture was allowed to dry on the plate. It was then covered with 1 microliter of α-cyano-4-hydroxycinnamic acid. At the same time, it was important to homogenize the sample and matrix (Jarolímková, 2017).

Unlike the analysis of bacteria, preprocessing of the yeast isolates was required to extract fungal proteins. The protein extraction method used to process yeast isolates for MALDI-TOF MS was adapted directly from established methods used to identify difficult bacterial isolates. Specifically, 1 to 5 colonies of an isolate were inactivated in 75% ethanol, pelleted, and then suspended in a 1:1 mixture of 70% formic acid and acetonitrile. The resulting supernatant was then analyzed by MALDI-TOF MS (Marklein et al., 2009; Bader et al., 2011; Dhiman et al., 2011). The results of the identifications are in Table 5.

This procedure aimed to cultivate individual isolates of technologically important microorganisms and preserve them using the lyophilization method. Separate cultivation of individual microbial isolates was performed based on the information obtained from the growth characteristics. Individual taxa were first pre-cultured in 250 mL Erlenmeyer flasks (100 mL medium volume; orbital stirring 120 rpm; 20 °C). The media and the culture times are shown in (Table 3). The pre-cultured cell suspension was examined microscopically (cell morphology, elimination of contamination) and centrifuged (10,000 rpm; 10 mins; 10 °C).

After separating the supernatant, the pellet was washed with saline solution and re-centrifuged (10,000 rpm; 10 mins; 10 °C) and resuspended in the pure culture medium. The prepared suspension served as the inoculum for the second cultivation stage, which was carried out in 2,000 mL Erlenmeyer flasks (1,000 mL medium volume; orbital stirring 100 rpm; 30 °C). The media and the culture times used for the individual isolates are shown in (Table 5).

After the cultivation was complete, the suspension was repeatedly centrifuged and washed as described in the previous step. The obtained biomass was mixed with cryoprotective medium and shock-frozen (70 °C; 24 h). The frozen suspension was then lyophilized. The viability of the obtained dehydrated biomass was then determined, and according to the qualitative and quantitative microbiological analysis (Table 2) and the cell viability in the lyophilisate, the 2018 Wine Microorganism Consortium was compiled.

Individual yeast isolates were initially cultured in 50 mL Erlenmeyer flasks (25 mL medium volume; orbital stirring 120 rpm; 30 °C). The media and the culture times used for the individual isolates are shown in (Table 5). In the obtained cell suspension, the cell density was determined by microscopic cell counting in a so-called Bürker chamber. The calculated amount of this suspension was then pipetted to a final concentration of 10⁸ cells.mL^-1 in a 250 mL Erlenmeyer flask (100 mL YPDm medium volume; without shaking; 25 °C). Fermentation was monitored by the gravimetric method, and weight loss due to the metabolic conversion of fermentable sugars to carbon dioxide and ethanol was observed.

Individual isolates were inoculated onto a BIGGY agar identification medium using a microbiological loop. Individual Petri dishes were statically cultured at 30 °C for 3 days. Based on the visual evaluation, the individual isolates were marked as low, medium, and high producers of H₂S.

BiGGY, Bismuth Sulphite Glucose Glycine Yeast Agar, is based on the formulation developed by Nickerson (1953) and mainly used for the isolation and presumptive identification of Candida species. In a study of sulphite reduction by yeasts, the ability of many yeasts to reduce a bismuthyl hydroxy polysulphite was noted. Growth on an acidic or neutral medium containing bismuth sulphite produced black colonies because of the extra-cellular reduction of the bismuth sulphite, to bismuth sulphide. The bismuth sulphite complex confers a high degree of selectivity to the medium, and most strains of bacteria are inhibited on BIGGY Agar. In this study, BIGGY agar was used as a simple and rapid method to compare the rate of H₂S production between pure yeast isolates.

Statistical analyses and figures were generated using Excel 2007 software packages (manufactured by Microsoft Office, USA) and Statistica 10 statistical software (Copyright © StatSoft). The Statistica 10 software was used to process growth curves data and create their line graphs.

The results of the microbiological analysis are shown in (Table 1). These data are comparable with the normal course of fermentation of the grape must. Based on the phenotypic analysis, a consortium was selected from the point of micro-sparkling.

The quantitative parameters of the individual taxa of the 2018 Wine Microorganism Consortium are given in (Table 2). The individual values in (Tables 1 and Table 2) were averaged from three measurements.

Through the application of microbiological techniques, the 2018 Wine Microorganism Consortium was obtained from the spontaneous batch, which was characterized in qualitative and quantitative terms.

The growth curve courses are shown in (Figures 1 and Figure 2), while the numerical parameters of the growth characteristics are shown in (Tables 3 and Tables 4). The growth characteristics of individual isolates were determined based on the growth curves of different types of media. These characteristics were used to assign culture media and culture times to individual isolates (Table 3).

To culture and lyophilise the 2018 Wine Microorganism Consortium, 30 g of the consortium was prepared and used for fermentation for 100 litres of must. The representation of the individual isolates can be seen in Table 4.

The growth curves, fermentation tests, and H₂S production results were used to inform the composition of the consortium so that the entire mixture exhibited the best fermentation capabilities, including the desired strains of lactic acid bacteria. Isolates Y03, Y06, and Y07 had the largest mass representation as characterized by a looser fermentation process but had the ability to ferment alcohol to 6.98, 9.37, and 7.20% vol. alc., respectively. These isolates showed low, medium, and high H₂S-producing ability. Isolates Y02 and Y05 had the second largest mass representation of the resulting consortium as characterized by high sugar fermentability up to an alcohol content of 13.28 and 13.30% vol. alc., respectively. The fermentation process was not gradual, however, which could negatively affect the quality of the wine. Both isolates showed mean H₂S production capacity. The isolate Y04 had a low mass representation. It showed the lowest sugar fermentability to an alcohol content of only 2.98 and had a medium ability to produce H₂S.

Table 5 contains the identification of individual isolates. The consortium consists of 2 strains of lactic acid bacteria and 7 strains of saccharomyces and non-saccharomyces yeast. The most abundant yeast strains of Consortium are Hanseniaspora uvarum, Zygosaccharomyces bailii and Candida sake.